Colony counts of proteolytic ruminal bacteria in the order of 1 x 10⁹ organisms per gram of whole rumen contents and total colony counts in the order of 2 to 3 x 10⁹ organisms per gram were obtained from rumen contents of cattle fed a maintenance ration of hay and grain. The proteolytic counts averaged 381 of the total counts. An anaerobic, differential medium characterizing proteolytic colonies by clear zones in an opaque skim milk suspension was utilized. Proteolytic isolates were assigned to the following taxa; Butyrivibrio fibrisolvens, Succinivibrio dextrinosolvens, Selenomonas ruminantium var. lactilyticas, Borrelia, Bacteroides (butyric acid-producing R•2 group of Bryant), and selenomonad-like organisms similar to B-385 group of Bryant. 1 A portion of the Ph.D. dissertation by the senior author. 2 Present address: Division of Natural Sciences, Susquehanna University, Selinsgrove, Pennsylvania. Proteolysis in the ruminal fermentation may benefit the host animal if the resulting products are later synthesized to digestible microbial proteins of higher biological value than the feed protein, or conversely this activity may be detrimental because of net protein loss. In spite of the nutritional significance of this activity the proteolytic ruminal bacteria have received relatively little attention as a physiological group. Studies have largely been restricted to observations of the degradation of gelatin or casein incidental to other studies of ruminal organisms. Gelatin proteolysis was reported among isolates from the rumen by Bryant (1951), Bryant and Burkley (1953a, 1953b, 1953c), Bryant and Small (1956a), Bryant et al. (1958a), Hamlin and Hungate (1956), Buhtanen and Gall (1953), Hungate (1957), and Hann et al. (1954). Bryant and Doetsch -- (1954) also reported isolates which attacked casein but not gelatin and Bryant (1956) reported a strain of Selenomonas ruminantium which digested casein but not gelatin. Bryant (1959) revealed the paucity of information on the proteolytic flora of the rumen in his excellent review of the bacteriology of the rumen. A casein medium designed for the isolation of proteolytic ruminal bacteria was described by Appleby (1955). Blackbum and Hobson (1960a) found proteolytic activity in all fractions of rumen contents (protozoa, large bacteria and small bacteria) and they initiated isolation of proteolytic bacteria from the ovine rumen (Blackburn and Hobson, 1960b). Fulghum (1958) described the development of two anaerobic, differential media for the isolation and enumeration of proteolytic ruminal bacteria, these media were developed through modification of the media of Hamlin and Hungate (1956) and ling and Smith (1955), and of the medium used by Donovan and Vincent (1955) for studying proteolytic organisms from milk. Fulghum (1958) found the optimum level of clarified rumen fluid added to these media to be 401. Colonies of proteolytic organisms in these media were characterized by clear zones in opaque skim milk or plant protein suspensions in the media. Plant proteins failed to maintain a uniform opacity and were therefore of limited value in delineating the proteolytic segment of the flora even though the plant proteins stimulated total counts by a factor of from three to five. Earlier Fulghum et al. (1958) reported that dispersion of whole rumen contents in anaerobic diluting fluid in a blendor increased total counts by a factor of four when compared with total counts obtained from rumen fluid samples which were diluted by shaking in anaerobic diluting fluid. Proteolytic counts were the same from both inocula. In later studies (Fulghum, 1958), proteolytic counts were also found to be increased by a factor of four when dispersed whole contents were compared with shake dilutions of rumen fluid. Proteolytic and total counts were found to be slightly higher in the dorsal sac of the rumen than in the ventral sac, although this phenomenon was variable with regard to time of sampling following feeding of animals. Similarly, the ratio of proteolytic to total counts varied at different times following feeding. The proteolytic flora remained constant while the total counts varied. The sequence of fluctuation was different in each individual animal. / Ph. D.